Telomeric Probes Research Articles

Karyotype heterogeneity in Philodendron s.l. (Araceae) revealed by chromosome mapping of rDNA loci.

Philodendron s.l. (Araceae) has been recently focus of taxonomic and phylogenetic studies, but karyotypic data are limited to chromosome numbers and a few published genome sizes. In this work, karyotypes of 34 species of Philodendron s.l. (29 species of Philodendron and five of Thaumatophyllum), ranging from 2n = 28 to 36 chromosomes, were analyzed by fluorescence in situ hybridization (FISH) with rDNA and telomeric probes, aiming to understand the evolution of the karyotype diversity of the group. Philodendron presented a high number variation of 35S rDNA, ranging from two to 16 sites, which were mostly in the terminal region of the short arms, with nine species presenting heteromorphisms. In the case of Thaumatophyllum species, we observed a considerably lower variation, which ranged from two to four terminal sites. The distribution of the 5S rDNA clusters was more conserved, with two sites for most species, being preferably located interstitially in the long chromosome arms. For the telomeric probe, while exclusively terminal sites were observed for P. giganteum (2n = 30) chromosomes, P. callosum (2n = 28) presented an interstitial distribution associated with satellite DNA. rDNA sites of the analyzed species of Philodendron s.l. species were randomly distributed considering the phylogenetic context, probably due to rapid evolution and great diversity of these genomes. The observed heteromorphisms suggest the accumulation of repetitive DNA in the genomes of some species and the occurrence of chromosomal rearrangements along the karyotype evolution of the group.

First cytogenetic analysis of lesser gymnures (Mammalia, Galericidae, Hylomys) from Vietnam.

Gymnures are an ancient group of small insectivorous mammals and are characterized by a controversial taxonomic status and the lack of a description of karyotypes for certain species. In this study, conventional cytogenetic techniques (Giemsa, CBG- and GTG-banding, Ag-NOR), CMA3-DAPI staining, and fluorescent in situ hybridization (FISH) with telomeric DNA probes were used to examine for the first time the karyotypes of lesser gymnures of group Hylomyssuillus Müller, 1840 from northern and southern Vietnam. All studied specimens had karyotypes with 2n=48, NFa=64. C-positive heterochromatic blocks existed in centromeric regions of 7 bi-armed autosomes and the submetacentric X chromosome. The Y chromosome is a C-positive and dot-like. The nucleolus organizer regions resided terminally on the short arms of 2 small bi-armed pairs. Positive signals at the telomeres of all chromosomes were revealed by FISH. CMA3-positive blocks were localized on the telomeric and pericentric regions of most bi-armed and acrocentric chromosomes. Despite the large genetic distances between Hylomys Müller, 1840, lesser gymnures from H.suillus-group from northern and southern Vietnam have similar karyotypic characteristics.

Cytogenetics description in Batrachoides surinamensis, (Batrachoididae: Batrachoidiformes): What does the estuary have to say?

The Batrachoididae (Batrachoidiformes) is a diverse fish family (84 species) of considerable medical interest, responsible for large numbers of injuries in fishermen and bathers in northern Brazil. Batrachoides surinamensis (the Pacuma toadfish) is the most common Batrachoididae species in the Amazon coastal zone. The capacity of this species to adapt to the dynamic environment of estuaries, its sedentary behavior, and benthic spawning all contribute to the interest in this fish as a model for the study of patterns of chromosomal diversification. The present study investigated the chromosomal features of this species through conventional (Giemsa, C-banding, and Ag-NOR) and molecular (FISH mapping of the 18S and 5S rRNA, and the telomeric sequences) cytogenetic approaches. This is the first description of the karyotype of this species, which has a diploid number (2n) of 46 chromosomes (6m+8sm + 20st+12a) and a fundamental number (FN) of 80. All the chromosomes presented C-banding in the pericentromeric region. The AgNOR/18S rRNA sites were observed exclusively on the short arms of pair 13, whereas multiple 5S rRNA sites were found, on pairs 7 (submetacentric) and 20 (acrocentric). The telomeric probes revealed interstitial telomeric sequences (ITSs) in metacentric pair 3, indicating the occurrence of chromosome fusion. The karyotype of B. surinamensis presents a number of derived karyotypic features, and is differentiated primarily by its pericentric inversions and the apparent occurrence of at least one chromosome fusion event. The collection of additional cytogenetic data on other Batrachoididae species, and other populations of B. surinamensis, will provide further insights into the role of estuarine environments in the chromosomal diversification of this fish group.

Chromosomal Abnormalities in Human Lymphocytes after Computed Tomography Scan Procedure.

The incidence of chromosomal abnormalities and cancer risk correlates well with the radiation dose after exposure to moderate- to high-dose ionizing radiation. However, the biological effects and health risks at less than 100 mGy, e.g., from computed tomography (CT) have not been ascertained. To investigate the biological effects of low-dose exposure from a CT procedure, we examined chromosomal aberrations, dicentric and ring chromosomes (dic+ring), in peripheral blood lymphocytes (PBLs), using FISH assays with telomere and centromere PNA probes. In 60 non-cancer patients exposed to CT scans, the numbers of dicentric and ring chromosomes were significantly increased with individual variation. The individual variations in the increment of dicentric and ring chromosomes after CT procedures were confirmed using PNA-FISH analysis of PBLs from 15 healthy volunteers after in vitro low-dose exposure using a 137Cs radiation device. These findings strongly suggest that appropriate medical use of low-dose radiation should consider individual differences in radiation sensitivity.

Evolution of trans-Andean endemic fishes of the genus Cheirodon (Teleostei: Characidae) are associated with chromosomal rearrangements

BackgroundAmong Neotropical fishes, the family Characidae is highly diverse and speciose and its taxonomy is not completely resolved. In Chile, the family is represented by five species, all within the genus Cheirodon, of which C. pisciculus, C. galusdae, C. kiliani, and C. australe are endemic, while C. interruptus is introduced. We compared chromosomal markers in order to appreciate the taxonomy and evolution of these trans-Andean fishes.ResultsThe specimens were photographed in stereomicroscope to observe the ventral protrusive teeth and cusps for morphological analysis and species identification. All of the species analysed had equally diploid chromosome number 2n = 50, with karyotypes dominated by high number of acrocentric chromosomes as compared to those of other members of Cheirodontinae. The distribution of heterochromatin was inconspicuous and was similar in all the species. The number of active NORs (nucleolus organizer regions) was polymorphic, with the greater number of them in C. kiliani and C. galusdae. The location of 5S and 18S rDNA ranged in number and position, showing two sites in different chromosomes. The fluorescent in situ hybridization with telomeric probe did not reveal interstitial sites in all analysed species.ConclusionsThe comparative analysis of karyotypes and morphological markers revealed a biogeographic pattern of distribution, with the species that occur in the southern region forming one group and those in central and northern Chile forming another.

Cytogenetics in Arctica islandica (Bivalvia, Arctidae): the Longest Lived Non-Colonial Metazoan.

Due to its extraordinary longevity and wide distribution, the ocean quahog Arctica islandica has become an important species model in both aging and environmental change research. Notwithstanding that, most genetic studies on ocean quahogs have been focused on fishery related, phylogeographic and phylogenetic aspects but nothing is known about their chromosomes. In this work, the chromosomes of the ocean quahog Arctica islandica were analysed by means of 4′,6-diamidino-2-phenylindole (DAPI)/propidium iodide (PI) staining and fluorescent in situ hybridization (FISH) with rDNA, histone gene and telomeric probes. Whilst both 5S rDNA and 45S rDNA were clustered at single subcentromeric locations on the long arms of chromosome pairs 2 and 12, respectively, histone gene clusters located on the short arms of chromosome pairs 7, 10 and 17. As happens with most bivalves, the location of the vertebrate type telomeric sequence clusters was restricted to chromosome ends. The knowledge of the karyotype can facilitate the anchoring of genomic sequences to specific chromosome pairs in this species.

RDNA와 말단소체 반복서열 탐침을 이용한 천마의 FISH 염색체 조성 분석

Background: Gastrodia elata Blume is a saprophytic perennial plant in the Orchidaceae family, because of its agricultural and medicinal effectiveness, researchers focus on its genome and chemical components. However, cytogenetic information based on the chromosome structure and composition to construct chromosomal backbone for genome sequencing research and for the development and breeding of plants is very limited.BR Methods and Results: We determined the metaphase chromosome composition of the G. elata genome by fluorescence in situ hybridization (FISH) using 5S and 45S rDNAs and telomeric repeat probes. The nuclear genome of G. elata was organized into 2 n = 36, with relatively small (2.71 - 5.50㎛)chromosomes that showed gradual decrease in size. Conglutination phenomenon was observed among the metaphase chromosomes, and it was distinguished from that in other plant metaphase chromosome spreads. One pair of signal was detected for each 5S and 45S rDNA in the pericentromeric region and interstitial region on the short arm of chromosomes 10 and 4, respectively, and telomeric DNA signals were detected in the terminal region of most chromosomes.BR Conclusions: To our knowledge, this is the first FISH chromosome composition result in G. elata and could be useful in more comprehensive molecular cytogenetic and genomic analyses as well as breeding programs of the medicinal plant G. elata.

Chromosomal Mapping of Repeat DNA in Bergiaria westermanni (Pimelodidae, Siluriformes): Localization of 45S rDNA in B Chromosomes

The occurrence of repetitive DNA in autosomes and B chromosomes of Bergiaria westermanni was examined using conventional and molecular cytogenetic techniques. This species exhibited 2n = 56 chromosomes, with intra- and interindividual variation in the number of heterochromatic B chromosomes (from 0 to 4). The 5S rDNA was localized in pairs 1 and 5, and histone probes (H1, H3, and H4) and U2 small nuclear RNA were syntenic with 5S rDNA in pair 5. Histone sequences were also located in chromosome pair 14. The (GATA)_n sequence was dispersed throughout the autosomes and B chromosomes, with clusters (microsatellite accumulation) in some chromosome regions. The telomeric probe revealed no signs of chromosomal rearrangements in the genome of B. westermanni. The 45S rDNA sites were detected in the terminal region of pair 27; these sites corresponded to a GC-rich heterochromatin block. In addition, 3 of the 4 B chromosomes also contained 45S rDNA copies. Silver nitrate staining in interphase nuclei provided indirect evidence of the expression of these rRNA genes in B chromosomes, indicating the probable origin of these elements. This report shows plasticity in the chromosomal localization of repeat DNA in B. westermanni and features a discussion of genomic diversification.

Dynamics of tandemly repeated DNA sequences during evolution of diploid and tetraploid botiid loaches (Teleostei: Cobitoidea: Botiidae).

Polyploidization has played an important role in the evolution of vertebrates, particularly at the base of Teleostei–an enormously successful ray-finned fish group with additional genome doublings on lower taxonomic levels. The investigation of post-polyploid genome dynamics might provide important clues about the evolution and ecology of respective species and can help to decipher the role of polyploidy per se on speciation. Few studies have attempted to investigate the dynamics of repetitive DNA sequences in the post-polyploid genome using molecular cytogenetic tools in fishes, though recent efforts demonstrated their usefulness. The demonstrably monophyletic freshwater loach family Botiidae, branching to evolutionary diploid and tetraploid lineages separated >25 Mya, offers a suited model group for comparing the long-term repetitive DNA evolution. For this, we integrated phylogenetic analyses with cytogenetical survey involving Giemsa- and Chromomycin A3 (CMA3)/DAPI stainings and fluorescence in situ hybridization with 5S/45S rDNA, U2 snDNA and telomeric probes in representative sample of 12 botiid species.The karyotypes of all diploids were composed of 2n = 50 chromosomes, while majority of tetraploids had 2n = 4x = 100, with only subtle interspecific karyotype differences. The exceptional karyotype of Botia dario (2n = 4x = 96) suggested centric fusions behind the 2n reduction. Variable patterns of FISH signals revealed cases of intraspecific polymorphisms, rDNA amplification, variable degree of correspondence with CMA3+ sites and almost no phylogenetic signal. In tetraploids, either additivity or loci gain/loss was recorded. Despite absence of classical interstitial telomeric sites, large blocks of interspersed rDNA/telomeric regions were found in diploids only.We uncovered different molecular drives of studied repetitive DNA classes within botiid genomes as well as the advanced stage of the re-diploidization process in tetraploids. Our results may contribute to link genomic approach with molecular cytogenetic analyses in addressing the origin and mechanism of this polyploidization event.

A Comparative Chromosome Mapping Study in Japanese Podismini Grasshoppers (Orthoptera: Acrididae: Melanoplinae)

In the present paper, karyotypes of 7 Japanese Podismini species, Anapodisma beybienkoi, Fruhstorferiola okinawaensis, Parapodisma caelestis, P. mikado, P. setouchiensis, P. tenryuensis, and Sinopodisma punctata (2n♂ = 21, all acrocentric), are described and compared on the basis of conventional (C-banding, DAPI/CMA₃-staining, Ag-NOR) and molecular (FISH with 18S rDNA and telomeric probes) cytogenetic staining methods. This is the first study to report karyotypes of A. beybienkoi and P. caelestis. Differential staining techniques showed karyotypic diversity in these species. The number of 18S rDNA signals ranged from 2 to 6, and the signals were located on the autosomes or sex chromosomes. In all species, clusters of rDNA coincided with Ag-NORs. Telomeric signals occurred at the chromosome ends at the pachytene stage and seldom at other stages of meiosis. Paracentromeric and some distal and interstitial blocks of constitutive heterochromatin were detected in the chromosomes of Anapodisma, Fruhstorferiola, and Parapodisma species. Staining with DAPI and CMA₃ revealed 2 groups of heterochromatin composition. In addition, intraspecific differences in the number of rDNA clusters and C-bands were observed within Parapodisma species. Based on the evidence of cytogenetic characteristics, the monophyly of Tonkinacridina cannot be supported.

Chromosome Karyotyping of Senna covesii and S. floribunda based on Triple?color FISH Mapping of rDNAs and Telomeric Repeats

The genus Senna in the family Fabaceae is distributed and cultivated around the world and has been used as sources of medicine and commercial goods. Molecular cytogenetic study based on FISH technique is useful for breeding and genomic research of the species. Triple-color FISH karyotype analyses were carried out to understand the genome structure of two Senna species, S. covesii (A. Gray) H. S. Irwin & Barneby and S. floribunda (Cav.) H. S. Irwin & Barneby using 5S rDNA, 45S rDNA, and telomeric repeat probes. Three and one pairs of 45S and 5S rDNA signals were detected, respectively, in S. covesii, whereas one pair each of 45S and 5S rDNA signals, were detected in S. floribunda. Telomeric signals appeared at all chromosome termini in both species. This result provides basic cytogenetic information which will be useful for future breeding and genomic research of Senna species.

Chromosome Painting in Tyrant Flycatchers Confirms a Set of Inversions Shared by Oscines and Suboscines (Aves, Passeriformes)

Tyrannidae is the largest family of Passeriformes in the Neotropical region. However, despite an interesting chromosomal diversity, there are only few cytogenetic studies of this family, and most of these are based on conventional cytogenetics. Hence, we analyzed here the chromosomal diversity and karyotypical evolution of this group by chromosome painting in 3 different species - Pitangus sulphuratus, Serpophaga subcristata, and Satrapa icterophrys - and make comparisons with previous data. In addition to chromosome painting with Gallus gallus (GGA) and Leucopternis albicollis (LAL) probes, karyotypes were analyzed by conventional staining, C-banding, and FISH with 18S rDNA and telomeric probes. Although this family is characterized by extensive chromosomal variation, we found similar karyotypes and diploid numbers ranging from 2n = 80 in P. sulphuratus to 2n = 82 in S. subcristata and S. icterophrys. Constitutive heterochromatin was located centromerically in all 3 species. Clusters of 18S rDNA were present in 1 pair of microchromosomes, except in S. subcristata, where 2 pairs of microchromosomes were labeled. No interstitial telomeric sequences were detected. GGA and LAL whole-chromosome probes revealed the occurrence of fissions and both paracentric and pericentric inversions commonly seen in other Passeriformes. In general terms, tyrants show the typical karyotype found in Passeriformes, suggesting that the observed rearrangements occurred before the division of the suborders Oscines and Suboscines.

Cytogenetic characterization and description of an X1X1X2X2/X1X2Y sex chromosome system in Collichthys lucidus (Richardson, 1844)

The chromosomes of spinyhead croaker Collichthys lucidus (Richardson, 1844) were characterized for the first time by fluorescence staining, self genomic in situ hybridization (self-GISH), and multicolor fluorescence in situ hybridization (FISH) with 18S rDNA, 5S rDNA and telomeric sequence probes. The female karyotype has exclusively 24 pairs of acrocentric chromosomes (2n=48a, NF=48), while the male one consists of 22 pairs of acrocentric chromosomes, 2 monosomic acrocentric chromosomes and a metacentric chromosome (2n=1m+46a, NF=48). The difference between female and male karyotypes indicates the presence of a sex chromosome of X1X1X2X2/X1X2Y type, where Y is the unique metacentric chromosome in the male karyotype. As revealed by FISH, 5S rDNA and 18S rDNA sites were mapped at syntenic position of the largest acrocentric chromosome (X1), and the short arms of the Y chromosome as well. An X1-chromosome specific interstitial telomeric signal (ITS) was detected overlapping the 5S rDNA sites. In addition, self-GISH revealed that the repetitive DNAs accumulated on all the putative sex chromosome. Chromosome fusion accompanied by a partial deletion in the ancestral karyotype (2n=48a) is hypothesized for the origin of such multiple sex chromosome system. The present study, as the first description of differentiated sex chromosome in family Sciaenidae, will give clues to the studies on the sex chromosome of other Sciaenids.

XX/XY sex chromosome system and chromosome markers in the snake eel Ophisurus serpens (Anguilliformes: Ophichtidae)

ABSTRACTWe report evidence of an XX/XY sex chromosome system in the snake eel Ophisurus serpens (Anguilliformes: Ophichthidae). We characterized the male and female karyotypes by C-, replication- and HaeIII-bandings. The 45S and 5S ribosomal gene families were located using dual fluorescence in situ hybridization, which showed that the 5S rDNA sites were present on the X chromosome, beside an autosome pair. FISH with a telomeric peptide nucleic acid probe enabled recognition of Interstitial Telomeric Sequences (ITSs), likely remnants of chromosomal rearrangements, in five chromosome pairs, including the rDNA-bearing ones. Possible mechanisms of the origin of sex chromosomes in this species are discussed, considering the presence of a sex-linked marker and ITSs.

Molecular phylogenetic reconstruction and localization of the (TTAGG)n telomeric repeats in the chromosomes of Acromyrmex striatus (Roger, 1863) suggests a lower ancestral karyotype for leafcutter ants (Hymenoptera).

Chromosome counts and karyotype characterization have proved to be important features of a genome. Chromosome changes during the diversification of ants might play an important role, given the diversity and success of Formicidae. Comparative karyotype analyses on ants have enriched and helped ant systematics. Among leafcutter ants, two major chromosome counts have been described, one frequent in Atta Fabricius, 1804 (2n = 22 in all Atta spp. whose karyotype is known) and the other frequent in Acromyrmex Mayr, 1865 (2n = 38 in the majority of species whose karyotype is known). The main exception is Acromyrmex striatus (Roger, 1863), which harbors a diploid chromosome set of 22. Here we describe the use of fluorescence in situ hybridization (FISH) with telomeric probes with (TTAGG)6 repeats to describe the telomere composition of A. striatus and to recover potential interstitial non-telomeric signals that may reflect fusion events during the evolution of leafcutter lineage from 38 to 22 chromosomes. Further, we reconstruct the ancestral chromosome numbers of the leafcutter clade based on a recently proposed molecular phylogenetic hypothesis and phylogenomic tree. Distinct signals have been observed in both extremities on the telomere chromosomes of A. striatus. Non-telomeric signals have not been retrieved in our analysis. It could be supposed that the low-numbered karyotype indeed represents the ancestral chromosome number of leafcutters. The phylogenetic reconstruction also recovered a low chromosome number from the diverse approaches implemented, suggesting that n = 11 is the most likely ancestral karyotype of the leafcutter ants and is a plesiomorphic feature shared between A. striatus and Atta spp.

Subtelomeric Rearrangements in Patients with Recurrent Miscarriage.

BackgroundThe subtelomeric rearrangements are increasingly being investigated in cases of idiopathic intellectual disabilities (ID) and congenital abnormalities (CA) but are also thought to be responsible for unexplained recurrent miscarriage (RM). Such rearrangements can go unnoticed through conventional cytogenetic techniques and are undetectable even with high-resolution molecular cytogenetic techniques such as array comparative genomic hybridization (aCGH), especially when DNA of the stillbirth or families are not available. The aim of the study is to evaluate the rate of subtelomeric rearrangements in patients with RM.Materials and MethodsIn this cross-sectional study, fluorescent in situ hybridization (FISH), based on ToTelVysion telomeric probes, was undertaken for 21 clinically normal couples exhibiting a “normal” karyotype with at least two abortions. Approximately 62% had RM with a history of stillbirth or CA/ID while the other 38% had only RM.ResultsFISH detected one cryptic rearrangement between chromosomes 3q and 4p in the female partner of a couple (III:4) [46,XX,ish t(3;4)(q28-,p16+;p16-,q28+)(D3S4559+,D3S4560-,D4S3359+; D3S4560+, D4S3359- ,D4S2930+)] who presented a history of RM and family history of ID and CA. Analysis of the other family members of the woman showed that her sisters (III:6 and III:11) and brother (III:8) were also carriers of the same subtelomeric translocation t(3;4)(q28;p16).ConclusionWe conclude that subtelomeric FISH should be undertaken in couples with RM especially those who not only have abortions but also have had at least one child with ID and/or CA, or other clinically recognizable syndromes. For balanced and cryptic anomalies, subtelomeric FISH still remains the most suitable and effective tool in characterising such chromosomal rearrangements in RM couples.

Abstract A43: Genetic and epigenetic disorders in one cohort of acute lymphoblastic leukemia

Abstract B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease. Approximately 60% of BCP-ALL present aberrations involving chromosome 21 (chr 21), including hyperdiploid (HeH), ETV6-RUNX1 fusion, and intrachromosomal amplification of the chromosome 21 (iAMP21). On the other hand, epigenetic mechanisms could regulate the transcription and induce the leukemogenesis. Polymorphisms in folate genes could influence in this aberrant methylation. This study aims to characterize the genetic and epigenetic profile of BCP-ALL with chr21 aberrations, as well as identify the epigenetic signatures of different ALL subgroups. A series of 373 BCP-ALL were selected for CNA analysis concerning the chromosome 21. Multiplex ligation probe amplification (MLPA, SALSA P327_A1, and P327_B1) was performed according to manufacturer instructions. FISH was performed using the LPH012 TEL/AML1 translocation, dual fusion probe, and centromere probes to the chr 4, 8, 10, 14, 17, 18, X, and Y (Cytocell, Cambridge, UK). Additionally, to the DNA from cases of BCP-ALL, controls and remission samples were modified with EZ DNA Methylation™ Kit (Zymo Research, Irvine, CA) and analyzed by the HumanMethylation450 Infinium Assay (Illumina, San Diego, CA). For the array validation and global (LINE-1) methylation we analyzed not just the samples with chr 21 gains, but also BCP-ALL from different subtypes using pyrosequencing. The MTHFR rs1801133 was identified by PCR-RFLP. The statistical analysis was performed using RStudio software with Bioconductor packages. We found evidence of gains in chr21 in 82 samples analyzed by MLPA. Most gains were verified by FISH and 11 samples had ≥5 RUNX1 signals. The centromere probes characterized 53 samples as HeH. The extra chr21 was confirmed in all cases in addition to other gains: chr4 (58%), 10 (57%), 14 (84%), 17 (53%), 18 (60%), X (86%), and Y (46%). Losses of the chr 17 (2%) and Y (4%) were also identified. Two BCP-ALL with ETV6/RUNX1 had an extra copy of the chr 21. One patient had iAMP21 identified by MLPA and confirmed by FISH with telomere probe targeted to chr13 and 21. The case vs. control analysis regarding the DNA methylation showed 5031 differentially methylated CpG sites (adjusted p <0.001). We observed that the methylation profiles of cases and controls were distinct, while the controls and remission samples had similar profiles. In the validation analysis, the ARID3A gene was hypermethylated in controls when compared with BCP-ALL and remission samples. The HeH group presents lower methylation for the ERG and ARID3A genes while the BCP-ALL with KTM2A rearrangements had a higher methylation level for ARID3A, PRDM9, and PRDM15 genes, the ETV6-RUNX1 had a higher ARID3A methylation and lastly, the TCF3-PBX1 had a higher significant PRDM15 methylation when compared against the other BCP-ALL subtypes. According to the LINE-1 methylation, the HeH group present a distinct profile of increased methylation level in comparison with the other BCP-ALL subtypes and healthy controls. Genetic susceptibility conferred by MTHFR rs1801133 has not contributed to methylation changes in settings. To discriminate the iAMP21 to HeH subgroups, it is recommended to use centromere and/or telomere FISH probes. The BCP-ALL presents a distinct global and gene-specific DNA methylation signature; these signatures are subtype-specific. Citation Format: Tállita Meciany Farias Vieira, Sheila Coelho Sores Lima, Franciane Gomes Andrade, Alython Araújo Chung-Filho, Claire Schwab, Christine Harrison, Maria do Socorro Pombo-de-Oliveira. Genetic and epigenetic disorders in one cohort of acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A43.

Fluorescence in situ Hybridization Karyotype Analysis of Seven Platycodon grandiflorum (Jacq.) A. DC. Cultivars

Platycodon grandiflorum has long been cultivated for its medicinal properties and economic value, prompting crop improvement programs that have spurred the development of new cultivars. However, limited cytogenetic information is available for this species, especially for different P. grandiflorum cultivars. Karyotype analysis via fluorescence in situ hybridization (FISH) provides essential information about chromosome count and the localization of DNA sequences, thus providing a chromosome-level understanding of genome structure. Here, we carried out triple-color FISH karyotype analysis of various P. grandiflorum (Jacq.) A. DC. cultivars using 5S and 45S ribosomal DNA (rDNA) and telomeric repeat probes. The karyotype is composed of 2n = 18 chromosomes consisting of seven pairs of metacentric chromosomes, one pair of submetacentric chromosomes, and one pair of subtelocentric chromosomes. The chromosome length ranges from 1.92 to 4.98 μm. One pair of 5S rDNA loci was observed on either the intercalary or pericentromeric regions of chromosome 3. All cultivars showed 45S rDNA signals on chromosome 9, and in two closely related cultivars, one additional pair of 5S rDNAs co-localized with these 45S rDNAs. Arabidopsis -type telomeric repeats were observed in most telomeric regions of the chromosomes in all seven cultivars. Our data lay the foundation for future cytogenetic studies of P. grandiflorum focusing on breeding and the elucidation of its genome structure.

Ploidy and Number of Chromosomes in the Alveolate Alga Chromera velia

Chromera velia is an alveolate alga which represents the closest known phototrophic relative to apicomplexan parasites. Although the nuclear, mitochondrial, and plastid genomes of this alga have been sequenced, the number of chromosomes and ploidy of C. velia are unknown. We explored ploidy in the vegetative cell, the predominant stage in cultures of Chromera, using the tyramide signal amplification-fluorescence in situ hybridization (TSA-FISH) in isolated nuclei of C. velia. Probes were derived from three single copy genes coding for 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) synthase and Topoisomerase II. Our results indicate that the vegetative cell of C. velia is haploid, as each probe produced a single fluorescent signal, although the possibility of diploidy with somatic pairing of homologous chromosomes cannot be completely excluded. Restriction analysis and hybridization with the telomere probe produced eight bands suggesting the presence of four chromosomes in haploid vegetative cells of C. velia. However, when the chromerid-specific telomere probe (TTTAGGG)4 was used for TSA-FISH, we consistently obtained a double signal. This may indicate that the four chromosomes are organized in clusters in interphase nuclei of C. velia, which is a chromosome organization similar to that of their apicomplexan relatives.

New Insights into Phasmatodea Chromosomes.

Currently, approximately 3000 species of stick insects are known; however, chromosome numbers, which range between 21 and 88, are known for only a few of these insects. Also, centromere banding staining (C-banding) patterns were described for fewer than 10 species, and fluorescence in situ hybridization (FISH) was applied exclusively in two Leptynia species. Interestingly, 10–25% of stick insects (Phasmatodea) are obligatory or facultative parthenogenetic. As clonal and/or bisexual reproduction can affect chromosomal evolution, stick insect karyotypes need to be studied more intensely. Chromosome preparation from embryos of five Phasmatodea species (Medauroidea extradentata, Sungaya inexpectata, Sipyloidea sipylus, Phaenopharos khaoyaiensis, and Peruphasma schultei) from four families were studied here by C-banding and FISH applying ribosomal deoxyribonucleic acid (rDNA) and telomeric repeat probes. For three species, data on chromosome numbers and structure were obtained here for the first time, i.e., S. inexpectata, P. khaoyaiensis, and P. schultei. Large C-positive regions enriched with rDNA were identified in all five studied, distantly related species. Some of these C-positive blocks were enriched for telomeric repeats, as well. Chromosomal evolution of stick insects is characterized by variations in chromosome numbers as well as transposition and amplification of repetitive DNA sequences. Here, the first steps were made towards identification of individual chromosomes in Phasmatodea.